The specific aim of this proposal is to define the biochemical mechanism by which insulin regulates the growth response in the H35 hepatoma cell line. In this cell line the growth response to insulin is mediated through the insulin receptor. Membranes from the cultured cells will be prepared following incubation of the cells under varying hormonal and pharmacological conditions which influence the growth of the cells. The membranes will be analyzed for the ability of insulin to stimulate the release of the chemical mediator. The mediator will be assayed by its ability to activate mitochondrial pyruvate dehydrogenase. Fractions containing the factor will also be examined for the ability to stimulate total, drug sensitive and specific gene transcription in isolated nuclei. In order to determine the nature of the mediator in these cells, it's physical and chemical properties will be compared with the chemical mediator released in response to insulin from rat liver plasma membranes. Finally, the sequence and structural homology of the related insulin-like growth factors suggests that these molecules act in a similar fashion. Membranes from the H35 cells and the cultured L6 myoblasts will be exposed to these related growth factors under conditions where they would interact with their specific receptors and analyzed for the release of chemical mediators similar to that observed with insulin. Once again, activation pyruvate dehydrogenase will be employed as assay for this mediator and then effects on transcription will be assessed with fractions containing factor. These will be characterized to determine whether the "putative" chemical mediators released in response to the insulin-like growth factors are similar to that released in response to insulin. The long range goals of the project are to define the biochemical events that occur following insulin binding to the receptor, including the release of a chemical mediator, its interaction with the control elements regulating the quiescent state of the cell, and the subsequent events involved in stimulating the transition through the cell cycle.